Unmasking Cell Replication

Reagents for Analyzing Cell Cycle and Replication in Mammalian Cells

Measuring a cell’s ability to proliferate is a fundamental method used in developmental and stem cell biology, cancer research, and drug discovery and toxicology. Several techniques are now available to efficiently and accurately measure the progression of a cell through the cell cycle. Premo™ FUCCI Cell Cycle Sensor is designed to provide an accurate and sensitive readout of cell cycle progression in individual cells or in a population of cells. This genetically encoded sensor is delivered by the efficient BacMam 2.0 technology, enabling cell cycle analysis in a wide range of cell types. Click-iT® EdU assays for imaging and flow cytometry platforms are useful for detecting cells transitioning into the DNA synthesis phase of the cell cycle and for distinguishing these S-phase cells from cells in other phases of the cell cycle.

Study Cell Cycle in Context With Premo™ FUCCI Sensor

Developed by Miyawaki and colleagues [1], Premo™ FUCCI Cell Cycle Sensor is based on two cell cycle–regulated proteins, geminin and Cdt1, fused to green (emGFP) and red (TagRFP) fluorescent proteins, respectively. Degradation of Cdt1 and geminin proteins are temporally regulated such that only Cdt1 is present in the G1 phase, both proteins are present during the G1/S transition, and only geminin is present during the S, G2, and M phases. As a result, nuclear fluorescence changes from red (G1) to green (S, G2, M), providing a simple, elegant technique to clearly monitor progression through the cell cycle (Figure 1).

The FUCCI system is based on BacMam 2.0 technology, which utilizes an insect virus (baculovirus) for efficient transduction and transient expression in mammalian cells. BacMam 2.0 greatly expands the utility of this popular gene delivery platform. Cell types previously not compatible with BacMam 1.0 (e.g., neurons) or those only poorly transduced (cryopreserved primary cells, CHO cells, and some stem cells) are now transduced quantitatively with a simple protocol. BacMam 2.0 also incorporates elements that improve transduction efficiency and expression levels: a pseudotyped capsid protein for more effective cell entry, as well as an enhanced CMV promoter and the woodchuck hepatitis post-transcriptional regulatory element (WPRE) to boost transcription levels.

Premo™ FUCCI Cell Cycle Sensor enables live-cell imaging of cell cycle progression and cell division, and it is also compatible with fixation and permeabilization treatments for subsequent immunocytochemical analysis. This cell cycle sensor is ready to use, and the procedure is easy—simply add Premo™ FUCCI reagents to your cells, incubate overnight for protein expression, and visualize using fluorescence microscopy or flow cytometry.

Figure 1. Visualization of cell cycle phases with Premo™ FUCCI Cell Cycle Sensor. (A) Progression through the cell cycle. Images of cells expressing thePremo™ FUCCI Cell Cycle Sensorwere acquired at the indicated time points over a 24-hour period. Initially, the cell in the center of the image transitions from green (G2/M phase) to red (G1 phase) to yellow (S phase). At approximately 5 hr 40 min, the cell at the top of the image migrates down before undergoing mitosis (green) and progressing into G1 phase (red). (B) U2OS cells expressing Premo™ FUCCI Cell Cycle Sensor (green, yellow, red) were fed 10 µM EdU, fixed, and labeled using the Click-iT® EdU Alexa Fluor® 647 Imaging Kit (purple) and an anti–α-tubulin antibody followed by an Alexa Fluor® 405 goat anti-mouse secondary antibody (blue). Click-iT® labeling (purple) can be seen in some of the S/G2 phase cells (green). Images were captured on a Zeiss LSM 710 confocal microscope.

The Click‑iT® EdU imaging and flow cytometry assays are novel alternatives to the bromodeoxyuridine (BrdU) assay. The nucleoside analog EdU (5-ethynyl-2′-deoxyuridine) is incorporated into nascent DNA in place of thymidine during active DNA synthesis [2]. Detection of incorporated EdU is based on the click reaction—a highly specific and efficient covalent reaction between the alkyne moiety in EdU and an azide coupled to an Alexa Fluor® or Pacific Blue™ fluorophore. The percentage of S-phase cells in the population can be determined using standard imaging techniques (Figure 1B), flow cytometry methods (Figure 2), or high-content screening analysis.

A key advantage of Click-iT® EdU labeling is that the small size of the fluorescent azide allows for efficient detection of the incorporated EdU using mild conditions that preserve antigen integrity and thus enable downstream antibody-based analyses. In contrast to the harsh acid and DNase treatments required in BrdU-based methods, standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT® detection reagent to gain access to the DNA.

Figure 2. Multiparameter cell cycle analysis using the Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Kit. HeLa cells growing in culture conditions were fed a 2-hour pulse of 10 µM EdU or vehicle, followed by fixation and permeabilization steps with buffers contained in the Click-iT® EdU flow cytometry kits. Cellular incorporation of EdU was detected by a click reaction with Alexa Fluor® 488 dye azide. Dual-parameter plots of Click-iT® EdU Alexa Fluor® 488 fluorescence and FxCycle™ Violet stain, which labels dsDNA in fixed cells, can be used to quantify cells in each phase of the cell cycle. (A) Cells that were fed EdU show a clear separation between cells in S phase and cells in the G0/G1 and G2/M phases. (B) Control cells.